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. 2009 Jun 8;4(6):e5833.
doi: 10.1371/journal.pone.0005833.

Multi-locus assortment (MLA) for transgene dispersal and elimination in mosquito populations

Jason L Rasgon  1
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Multi-locus assortment (MLA) for transgene dispersal and elimination in mosquito populations

Jason L Rasgon. PLoS One. .
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Abstract

Background: Replacement of wild-type mosquito populations with genetically modified versions is being explored as a potential strategy to control vector-borne diseases. Due to lower expected relative fitness of transgenic individuals, transgenes must be driven into populations for these scenarios to be successful. Several gene drive mechanisms exist in a theoretical sense but none are currently workable in mosquitoes. Even if strategies were workable, it would be very difficult to recall released transgenes in the event of unforeseen consequences. What is needed is a way to test transgenes in the field for feasibility, efficacy and safety prior to releasing an active drive mechanism.

Methodology/principal findings: We outline a method, termed Multi-locus assortment (MLA), to spread transgenes into vector populations by the release of genetically-modified mosquitoes carrying multiple stable transgene inserts. Simulations indicate that [1] insects do not have to carry transgenes at more than 4 loci, [2] transgenes can be maintained at high levels by sequential small releases, the frequency of which depends on the construct fitness cost, and [3] in the case of unforeseen negative non-target effects, transgenes can be eliminated from the population by halting transgenic releases and/or mass releases of wild-type insects. We also discuss potential methods to create MLA mosquito strains in the laboratory.

Conclusions/significance: While not as efficient as active drive mechanisms, MLA has other advantages: [1] MLA strains can be constructed for some mosquito species with currently-available technology, [2] MLA will allow the ecological components of transgenic mosquito releases to be tested before actual gene drive mechanisms are ready to be deployed, [3] since MLA is not self-propagating, the risk of an accidental premature release into nature is minimized, and [4] in the case that active gene drive mechanisms prove impossible to develop, the MLA approach can be used as a back-up transgene dispersal mechanism for disease control efforts in some systems.

Conflict of interest statement

Competing Interests: The author has declared that no competing interests exist.

Figures

Figure 1. Crossing scheme to create an MLA mosquito strain.
A: Each transgenic line (A–D) is outcrossed to one of two wild-type backgrounds (denoted by colors). The final 8 outcrossed transgenic lines are made homozygous. B: The 8 outcrossed transgenic lines are specifically crossed and offspring of the correct genotype selected each generation to construct the MLA strain. Correct genotyping of offspring can potentially be automated in a high-throughput manner (see text and Figure 8). C: Hypothetical locations of each transgenic locus so that all 4 loci assort independently.
Figure 2. Single mass release.
Changes in transgene frequency after a single 30% release of mosquitoes homozygous for a transgene at 4 unlinked loci (8 copies/genome). Thick solid line: No fitness cost (F = 1.0), thin solid line: 1% fitness cost/insert (F = 0.99), thin dotted line: 5% fitness cost/insert (F = 0.95).
Figure 3. Continuous minimal release.
Changes in transgene frequency with 1% continuous release rate per generation of mosquitoes homozygous for a transgene at 4 unlinked loci (8 copies/genome). Thick solid line: no fitness cost (F = 1.0), thin solid line: 1% fitness cost/insert (F = 0.99), thick dotted line: 5% fitness cost/insert (F = 0.95), thin dotted line: 10% fitness cost/insert (F = 0.9).
Figure 4. Continuous moderate release.
Changes in transgene frequency with a 5% continuous release rate per generation of mosquitoes homozygous for a transgene at 4 unlinked loci (8 copies/genome). Thick solid line: no fitness cost (F = 1.0), thin solid line: 1% fitness cost/insert (F = 0.99), thick dotted line: 5% fitness cost/insert (F = 0.95), thin dotted line: 10% fitness cost/insert (F = 0.9).
Figure 5. Discontinuous release.
Changes in transgene frequency with a 5% release every 10 generations of mosquitoes homozygous for a transgene at 4 unlinked loci (8 copies/genome). Thick solid line: no fitness cost (F = 1.0), thin solid line: 1% fitness cost/insert (F = 0.99), thin dotted line: 5% fitness cost/insert (F = 0.95).
Figure 6. Passive transgene elimination.
Changes in transgene frequency with a 5% discontinuous release every 10 generations of mosquitoes homozygous for a transgene at 4 unlinked loci (8 copies/genome). Releases are halted after the fourth release. If transgenes are costly, selection eliminates the construct from the population. Thick solid line: no fitness cost (F = 1.0), thin solid line: 1% fitness cost/insert (F = 0.99), thin dotted line: 5% fitness cost/insert (F = 0.95).
Figure 7. Active elimination of a neutral transgene (F = 1.0). 4-locus homozygous mosquitoes are initially released at a 5% frequency every 10 generations.
At generation 40, transgenic releases are halted and the transgene eliminated by release of wild-type mosquitoes. Thick solid line: mass 99∶1 wild-type release, Thin solid line: 5 “burst releases” of wild-type at a rate of 20% for 5 generations separated by 5 generations, thin dotted line: continuous 5% wild-type release every generation.
Figure 8. Potential strategy using a self-assembling fluorescent reporter protein to visually identify mosquitoes carrying 1 vs. 2 inserts at a specific genomic phi C31 location.
Mosquitoes heterozygous for each individual transgene (identified by constitutive DsRED fluorescence) are crossed. F1 mosquitoes inheriting a transgene copy from each parent exhibit eye-specific (3xP3) GFP expression. When GFP-expressing F1 mosquitoes are crossed, all subsequent generations will be homozygous for the effector transgene. CP = bloodmeal-induced carboxypeptidase promoter, aC5 = constitutive actin5C promoter, T = terminator.
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